JPS6127038B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for culturing aerobic microorganisms.

The industrial cultivation of microorganisms is of increasing interest. In fact, the cultivation of microorganisms can be used to obtain the microorganism itself, for example to produce compounds secreted by the microorganism such as enzymes, pigments or toxins, to produce metabolic products such as antibiotics, vitamins or amino acids. or make it possible to biochemically transform certain limited compounds, as is the case, for example, during the industrial production of citric acid.

Furthermore, the use of certain microorganisms as protein sources for animal or human food represents a possible answer to the world's food resource crisis.

Microorganisms have an advantage over plants and animals commonly used for food, especially in that they have high growth rates. This much higher productivity could considerably increase the amount of food available.

Various methods have already been perfected for feeding and nourishing microorganisms with a wide variety of substrates and for extracting protein concentrates that can be used as food from the biomass thus obtained.

Most microorganisms used for this purpose are aerobic microorganisms. Therefore, in order to ensure the growth of these microorganisms, it is necessary to supply air or oxygen to the culture device. However, the solubility of oxygen in water is extremely low, so the growth rate of microorganisms is often limited by the rate of oxygen dissolution in water.

To overcome this drawback, British Patent No. 861,784 filed on January 10, 1958 by Armour suggests using an aqueous hydrogen peroxide solution as the oxygen source. The substrates used are those currently used in microbial cultivation, ie carbohydrates (molasses, peptones, glucose, lactose) or protein-rich products. However, these products have major drawbacks which make them even less interesting to use as substrates from an economic point of view.
In fact, it is a manufactured product, some of which is directly suitable as animal or human food without modification. On the other hand, some of these products are agricultural by-products and due to this fact require complex pretreatment before they can be used as substrates.

In another context, economic reasons have turned towards using substrates consisting of much simpler molecules, such as those derived from petroleum products. In British Patent No. 914,568 filed August 16, 1961 to British Petroleum, it is proposed to grow microorganisms in the presence of oxygen and a hydrocarbon such as kerosene. Extending this process to significantly lighter substrates such as methane, methanol, and mixtures of hydrogen and carbon monoxide or carbon dioxide poses major problems due to the flammability of the mixtures of these products with oxygen.

The applicant has invented a method that does not have the disadvantages of the above methods.

The present invention relates to a method for culturing aerobic microorganisms in a medium containing at least one oxygen source, at least one carbon source, and at least one hydrogen source, provided that the oxygen generated from the decomposition of hydrogen peroxide is used as the oxygen source and at least one highly flammable product is used as the carbon and hydrogen source.

Generally, the highly flammable products used according to the invention are selected from aliphatic monohydric alcohols containing 1 to 4 carbon atoms, mixtures of hydrogen with carbon monoxide and carbon dioxide. Highly flammable product means a product whose flash point is at or below 30°C.

Best results are obtained with methanol and mixtures of hydrogen and carbon monoxide or carbon dioxide. The mixture of hydrogen and carbon monoxide contains varying amounts of hydrogen, generally between 30 and 99% by volume, preferably between 60 and 95%.
Contains % hydrogen by volume. Mixtures of hydrogen and carbon dioxide contain varying amounts of hydrogen, typically between 40 and 99% by volume.
preferably 65 to 99% by volume of hydrogen.

By medium is meant a medium in which microorganisms grow, a medium in which microorganisms propagate, or a medium in which both operations are carried out simultaneously.

Generally, the culture medium is a liquid in which the microorganisms are dispersed. In this method, it is particularly preferable to decompose hydrogen peroxide in a microbial culture medium and therefore in the presence of microorganisms.

The decomposition of hydrogen peroxide is carried out with the aid of at least one catalyst that favors the decomposition of hydrogen peroxide.

This catalyst is either a regular component of the medium or an external product added to the regular medium.

Therefore, it is not always necessary to add an external catalyst when the microorganism contains products that are advantageous for the decomposition of hydrogen peroxide. This is the case, for example, in certain catalase-rich bacteria and certain fungi. However, the addition of small amounts of catalyst is not excluded. The same is true if one of the nutritional components normally present in the culture medium catalyzes the decomposition of hydrogen peroxide.

When the microbial medium components do not contain any compounds that catalyze the decomposition of hydrogen peroxide, one or more catalysts are added to the medium.

Examples of hydrogen peroxide decomposition catalysts include, for example, WC Schum, CN Satterfield, RL Wentworth, "Hydrogen Peroxide", ACS Monograph Series, No. 128, Reinhold Publishing Co., Ltd., New York, 1955, 467-501.
Shown on page. Organic and biological compounds are generally suitable. Among these, catalase and peroxidase, which are known to have high activity, are preferably used.

The amount of hydrogen peroxide used must be determined based on the maximum decomposition rate of hydrogen peroxide that can be obtained under the culture conditions, so as to avoid the presence of excess hydrogen peroxide in the culture medium.

This maximum permissible rate is highly dependent on the nature of the catalyst.

If the catalyst used is sufficiently effective and the amount of oxygen produced is higher than that required for microbial growth, the oxygen requirements of the microorganisms are met in such a way as to meet the above requirements without unduly exceeding the oxygen requirements of the microorganisms. Therefore, it is necessary to determine the amount of hydrogen peroxide to be introduced. The oxygen requirements of these microorganisms are highly dependent on the type of microorganism, its concentration in the medium, and the substrate used as a nutritional element.

The amount of hydrogen peroxide introduced into the culture medium per unit time is usually per mg of dry microorganisms formed per unit time.
It varies from 10 to 10,000 micromoles. The nature of the microbial species used may require the use of smaller or larger amounts of hydrogen peroxide. The use of carbon and hydrogen rich and oxygen poor substrates means more oxygen depletion.

Hydrogen peroxide is generally used in the form of a solution in a solvent compatible with the hydrogen peroxide and the culture medium. Water is generally used as a solvent. Solutions of significantly different concentrations can be used, but generally no more than 10% by weight, preferably 0.005
Preferably, a dilute solution containing ~8% by weight hydrogen peroxide is used. It is clear that very low concentrations require large equipment. High concentrations are undesirable. It is necessary to avoid the risk of local accumulation of hydrogen peroxide, which could lead to the death of microorganisms in case of insufficient stirring.

Preferably, hydrogen peroxide is introduced directly into the medium. Other methods can also be used in which nascent oxygen derived from the decomposition of hydrogen peroxide can be contacted with the medium.

For example, a hydrogen peroxide solution can be introduced into a nutrient medium, usually an aqueous solution, and all of it can be delivered directly to the medium. Other methods can also be used.

The method is applicable to the cultivation of all obligate or facultative aerobic microorganisms capable of assimilating the substrates that can be used according to the invention. Such microorganisms are chemotrophic because they derive the energy required for biosynthesis from fermentation reactions of organic compounds or from the oxidation of organic or inorganic compounds.

The method that is the object of the invention is preferably applied to heterotrophic (chemo-ganotrophic) microorganisms. Among these are a large number of bacteria, such as most pseudomonales, actinobacteria, a large number of fungi, such as most phycophycetes, ascomycetes, especially yeasts or yeasts, protozoa, and different types of animal cells.

This method is particularly well applicable to the cultivation of unicellular microorganisms.

The following types of bacteria can be pointed out as industrially interesting bacteria for which the present method is applicable. Acetobacter, acetomonas, arthrobacter, breviba
-cterium), Corynebacteria, Hydrogenomonas, Coccus, Mycobacterium, Nocardia, Pseudomonas, Streptomyces, Vibrio, etc. As fungi or yeasts, mention may be made in particular of the following types: Aspergillus, Candida, cryptococcoid-ees, Penicillium, Satucharomyces, torulopsis, etc.

It is clear that the type of microorganism to be cultivated is directly related to the nature of the substrate to be used. Therefore, when attempting to use methane as a substrate for protein production, it is advantageous to use bacterial microorganisms such as Pseudomonas. When the substrate is methanol, best results are obtained with yeast of the Candida type or bacteria of the Pseudomonas type. Best results are obtained with bacteria of the Hydrogenomonas type when the substrate is a mixture of hydrogen and carbon monoxide or carbon dioxide.

The nutrient media in which microbial cultures occur are quite complex and variable. Apart from the substrate and oxygen, the medium contains a number of nutritive minerals (substances) including elements such as nitrogen, potassium, calcium, magnesium, sulfur, phosphorus, and for example zinc, iron,
Contains small amounts of other trace elements essential for development such as copper and cobalt.

The nature of the microorganism also determines the temperature and pH at which the culture is carried out. Generally, the optimal culture temperature for many bacteria is 37°C.
The temperature for many fungi is around 28°C. However, certain microorganisms require lower or higher culture temperatures. The pH value usually varies between 4 and 8, with bacteria growing optimally around 7 and fungi between 4 and 8.
Requires a pH of 5. PH of the medium during cultivation of microorganisms
When the formation of various products that tend to change the PH is observed, it is often necessary to add to the medium buffers or other agents capable of maintaining the PH at its optimum value.

The method can be carried out continuously or discontinuously.
Various types of equipment can be used for this purpose. Agitation means and temperature control means are generally provided to avoid increases in temperature that could kill the culture.

This method allows microorganisms to be cultured with excellent growth rates. This method also avoids the risk of ignition of the substrate used.

The following example illustrates remarkable results obtained with one implementation of the invention, but is not intended to limit the invention.

The following examples were carried out at the request of the applicant at the Institute of Microbiology, University of Götztingen, Griesebach Street 8.
3400 Götztingen, carried out by Dr. Bernhard Sink under the supervision of Professor HG Schlegel, director of West Germany.

Examples 1, 2, and 3 were carried out using methanol as the substrate, and Example 4 was carried out using a mixture of hydrogen and carbon dioxide as the substrate.

Example 1 Inorganic compounds, DIFCO yeast extract 0.2%, methanol 0.5%, biotin 0.1 mg/,
30 ml of an aqueous solution containing 1.0 mg/ml of thiamine and 0.09 ml of catalase, or 90,000 international units/ml, was placed in a wide-mouth Erlenmeyer flask.

The inorganic element content per aqueous solution was as follows.

NH ₄ C 1g Na ₂ HPO ₄・12H ₂ O 9.0g KH ₂ PO ₄ 1.5g MgSO ₄・7H ₂ O 0.2g FeNH ₄ citrate 5mg CaC ₂・2H ₂ O 10mg Measured at 436 nm at the initial stage of suspension The medium was injected with Candida biogenii type yeast in an amount such that the optical density was 0.7.

An enzyme-free nitrogen stream was passed through the space of the culture vessel.

The temperature of the culture was maintained at 30°C by a thermostat and the medium was agitated with a stirrer at 100 rpm.

A 0.3% aqueous hydrogen peroxide solution was introduced into the medium at a rate of 2.5 ml/hour (test 1) and 5 ml/hour (test 2), respectively. An oxygen electrode immersed in the medium could control the free oxygen content.

Samples were taken from the culture medium every hour and the increase in microbial weight was calculated by measuring the optical density of the samples.

In both tests, the time required for the weight of the microorganisms to double was 3.5 hours. Exponential growth continued for 7 hours. The results obtained are shown in Table 1 (curves 1 and 2). The horizontal axis x represents time expressed in hours, and the vertical axis y represents optical density.

For comparison, all other conditions were tested in Tests 1 and 2 without introducing hydrogen peroxide and catalase.
The test (Test 3R) was carried out in the same manner as the test.

No microbial growth was observed. The obtained results are shown in the first round (curve 3R).

For comparison, another test (test 4R) was conducted without introducing hydrogen peroxide and catalase.
An air stream was passed through the space of the culture vessel at a rate of 0.5/min. Other conditions were the same as Tests 1, 2, and 3R. The results obtained are shown in FIG. 1 (curve 4R). Examination of the test shown in Figure 1 shows that the growth of Candida biogenii is unchanged when hydrogen peroxide is used in place of oxygen.

Example 2 200 ml of an aqueous solution containing the same proportions of the same inorganic compounds as in Example 1, 0.5% methanol, 0.1 mg of biotin, 1.0 mg of thiamine, 0.63 ml of catalase, and 0.2% of Difco yeast extract was placed in a wide-mouth Erlenmeyer flask. Ta.

Candida biogenii yeast was injected into the medium at a rate such that the initial optical density of the suspension was 3.9 as measured at 436 nm.

An enzyme-free nitrogen stream was passed through the space of the culture vessel.

The culture temperature was maintained at 30°C using a thermostat, and the culture medium was stirred using a stirrer rotating at 800 rpm.

A 4% aqueous hydrogen peroxide solution was introduced into the medium at a rate of 3.8 ml/hour and finally 5.4 ml/hour (Test 5). The oxygen electrode immersed in the medium was able to maintain the free oxygen content at 2 ml of oxygen/oxygen.

Hourly, the increase in the weight of microorganisms was checked as in Example 1.

How long does it take for the weight of the microorganism to double?
It took 4.8 hours. The results obtained are shown in FIG. 2, where x and y have the same definitions as in FIG. 1 (Test 5).

Test 5 in the absence of hydrogen peroxide and catalase under nitrogen flow (test 6R) and air flow (test 7R), respectively.
Two comparative tests were conducted using the same medium. The results obtained are shown by curves 6R and 7R in FIG. 2, respectively.

An examination of the results shown in Figure 2 shows that hydrogen peroxide is only slightly more effective than oxygen in terms of microbial growth.

Example 3 200 ml of an aqueous solution containing the same proportions of the same inorganic compounds as in Example 1, 1.0% methanol, 0.1 mg of biotin, and 0.63 ml of catalase was placed in a wide-mouth Erlenmeyer flask.

Pseudomonas of the Finn strain was introduced into the culture medium. The operating conditions were the same as in Example 2 (Test 5). A 4% aqueous hydrogen peroxide solution was introduced into the medium at a rate of 3.5 ml/hour (Test 8). The oxygen electrode immersed in the medium was able to maintain the free oxygen content at 2 mg/oxygen.

It took about 5 hours for the weight of the microorganisms to double. The results obtained are shown in FIG. 3, where x and y have the same definitions as in FIG.

Two comparative tests were carried out in the same medium as in test 8 in the absence of hydrogen peroxide and catalase, under nitrogen flow (test 9R) and air flow (test 10R), respectively. The results obtained are shown by curves 9R and 10R in FIG. 3, respectively.

An examination of the results shown in Figure 3 shows that hydrogen peroxide is slightly more effective than oxygen in terms of microbial growth.

Example 4 200 ml of an aqueous solution containing the same proportions of the same mineral compounds as in Example 1 and 0.45 ml of catalase were placed in a wide-mouth Erlenmeyer flask.

A bacterial strain of Alcaligenes eutrophus (=hydrogenomonas) H16 mutant PHB-4 was injected into the medium.

A gas stream containing 90% hydrogen and 10% carbon dioxide was passed through the space of the culture vessel.

The culture temperature was maintained at 30°C using a thermostat, and the culture medium was stirred using a stirrer rotating at 800 rpm.

A 4.8% hydrogen peroxide solution was introduced into the medium at a rate of 2 ml/hour (Test 11). An oxygen electrode immersed in the medium could regulate the free oxygen content.

Samples of the medium were taken hourly and the increase in microbial weight was calculated by measuring the optical density of the samples.

The time required for the weight of microorganisms to double is approximately 5
It was time. The obtained results are shown in curve 11 in Figure 4.
Indicated by However, x and y have the same definitions as in FIG.

Test 11 in the absence of hydrogen peroxide and catalase under a gas flow containing 70% hydrogen, 10% carbon dioxide, and 20% oxygen
A comparative test (Test 12R) was conducted using the same medium.
The results obtained are shown by curve 12R in FIG.

An examination of the results shown in Figure 4 shows that almost the same results can be obtained when hydrogen peroxide is used instead of oxygen in culturing Alcaligenes eutrophus H16PHB-4.

[Brief explanation of the drawing]

1 to 4 are diagrams showing the relationship between the optical density and time obtained in Examples 1 to 4, respectively.

Claims (1)

[Claims] 1. A saturated solution containing 1 to 4 carbon atoms and using oxygen generated by the decomposition of hydrogen peroxide as an oxygen source in an amount lower than the amount that would kill aerobic microorganisms in the culture medium. At least one oxygen source, characterized in that at least one highly flammable product selected from aliphatic monohydric alcohols and mixtures of hydrogen and carbon monoxide or carbon dioxide is used as carbon and hydrogen source. , a method for culturing aerobic microorganisms in a medium comprising at least one carbon source and at least one hydrogen source. 2. The method of claim 1, wherein the carbon and hydrogen sources are saturated aliphatic monohydric alcohols containing 1 to 4 carbon atoms. 3. The method according to claim 2, wherein the carbon and hydrogen sources are methanol. 4. The method of claim 1, wherein the carbon and hydrogen source is a mixture of hydrogen and carbon dioxide containing 40 to 99% by volume of hydrogen. 5. The method according to any one of claims 1 to 4, wherein hydrogen peroxide is decomposed in a medium. 6. The method according to claim 5, wherein hydrogen peroxide is added directly to the culture medium in the form of an aqueous solution. 7. The method according to claim 6, wherein the aqueous solution contains 10% by weight or less of hydrogen peroxide. 8. Claims 1 to 8, in which hydrogen peroxide is decomposed with the help of a catalyst that promotes the decomposition of hydrogen peroxide.
The method described in any one of paragraph 7.